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Diss Factsheets

Ecotoxicological information

Toxicity to other aquatic organisms

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Administrative data

toxicity to other aquatic vertebrates
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Study design and performance followed established protocols for microcosm systems. Control treatments were included for all statistical comparisons and characteristics of sediments and water composition (background and treatments) were analytically verified. study conditions less relevant for EU waters.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: The study design and performance was based on SETAC workshop discussions in Potsdam (EWOFFT: Hill et al. 1994), Lacanau (HARAP: Campbell et al. 1998), and Schmallenberg (CLASSIC: Giddings et al. 2002), which are summarized guidance documents.
Principles of method if other than guideline:
In addition, the refinements to existing protocols for application to a microcosm using a metal, methods followed those used by Schafers (2001).
Campbell PJ, Arnold DJS, Brock TCM, Grandy NJ, Heger W, Heimbach F, Maund SJ, Streloke M (eds.). 1999. Guidance document on higher-tier aquatic risk assessment for pesticides (HARAP). From the SETAC-Europe/OECD/EC workshop, Lacanau Océan, France, April 19-22, 1998. Society of Environmental Toxicology and Chemistry (SETAC), Pensacola Fl, USA.
Giddings JM, Brock TCM, Heger W, Heimbach F, Maund S, Norman S, Ratte HT, Schäfers C, Streloke M (eds.). 2002. Community-Level Aquatic Systems Studies – Interpretation Criteria (CLASSIC). Society of Environmental Toxicology and Chemistry (SETAC), Pensacola Fl, USA.
Hill IR, Heimbach F, Leeuwangh P, Matthiessen P (eds.). 1994. Freshwater Field Tests for Hazard Assessment of Chemicals. Lewis Publ., Boca Raton, FL, 503-513 European Workshop on Freshwater Field Tests (EWOFFT). Summary and recommendations report. Potsdam, Germany.
Schafers C. 2001. Community-level study with copper in aquatic microcosms. Submitted by Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany to International Copper Assoc. 109 pp
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc chloride
EC Number:
EC Name:
Zinc chloride
Cas Number:
Molecular formula:
zinc dichloride

Sampling and analysis

Analytical monitoring:
Details on sampling:
Water samples were collected every two weeks during the pre-treatment phase and exposure phase for analysis of major anions (Cl, SO4, PO4) and cations (Ca, Mg, Na, K), total and dissolved Zn, dissolved organic carbon (DOC) and total organic carbon (TOC), turbidity, total suspended (TSS) and total dissolved solids (TDS), hardness, alkalinity, and ammonia concentrations. Water samples were collected using a PVC sampler from a composite of five samples from each tank for all analyses.
Biological measurements including zooplankton species and abundance and phytoplankton species, abundance and chlorophyll a content, were taken from a composite sample from each replicate. Zooplankton samples were concentrated from 5 L to 100 mL, whereas phytoplankton samples were concentrated from 1 L to 100 mL. Samples were preserved with 2 mL of Lugol’s solution. Periphyton samples for chlorophyll a analysis were collected from floating artificial periphyton samplers (25 x 75 mm glass slides) every two weeks. Slides were then scraped and either preserved in Lugol solution or frozen until chlorophyll a analysis. Chlorophyll a analysis was used to estimate biomass using the fluorometric method. Additionally, primary productivity was determined every two weeks using an oxygen method, i.e., dissolved oxygen concentrations were measured using a titration method and differences in concentration between dark bottles (i.e., no photosynthesis) and light bottles (i.e., photosynthesis occurring) were used for estimating primary productivity.

Test solutions

Details on test solutions:
The study included two phases; base line and exposure. Baseline was initiated by randomly loading sediment (34 kg wet weight) to microcosm tanks. After approximately seven weeks of equilibration (baseline), the microcosm system was spiked with Zn to achieve nominal dissolved concentrations (approximately total to dissolved ratio was 4:1). Volume of spiked solutions for each microcosm tank was 10L. Spiked solutions were prepared from a concentrated stock ZnCl2. Spiked solutions were manually added to each microcosm tank using 3-L polyethylene containers. Spiked water was slowly and evenly distributed throughout the tanks to maximize mixing of microcosm and Zn spiked water. Water samples were taken from each tank for analysis of total and dissolved Zn concentrations at 1 day, 2 days, and 4 days after spiking. The system was respiked with Zn approximately every 4 days to obtain desired dissolved Zn concentrations. Due to variation in Zn concentrations between replicates, tanks were individually spiked with varying concentrations to achieve desired concentrations.

Test organisms

Test organisms (species):
other: microcosm/mesocosm
Details on test organisms:
Plankton communities were initially introduced into microcosm tanks from the water sample taken from the Everglades and then subsequently from a natural lake at William Country Club in North Miami, Florida, U.S.A. Water samples used for inoculation were analyzed for water chemistry as well as phytoplankton and zooplankton species identification. Benthic macro-invertebrate communities were not amended from what was established based on original sediment collection.

Study design

Test type:
Water media type:
Total exposure duration:
14 wk

Test conditions

49-73 mg CaCO3/l
Test temperature:
Dissolved oxygen:
5.6-8.7 mg/l
Nominal and measured concentrations:
five nominal treatment concentrations of zinc (8, 20, 40, 80, and 160 μg/L dissolved Zn) and a control. Measured concentrations as reported.
Details on test conditions:
Three replicates were used for each treatment. Microcosm tanks were 0.79-m3 round plastic tanks (0.76 m depth x 1.22 m diameter). The tanks were spaced 0.5 m apart and were randomly assigned for each treatment replicate. All microcosm tanks were under a clear plastic canopy to prevent rain from getting into the tanks, but allowing for natural light to reach the tanks.

Results and discussion

Effect concentrationsopen allclose all
14 wk
Dose descriptor:
Effect conc.:
14 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
Basis for effect:
other: chlorophyta eveness
14 wk
Dose descriptor:
Effect conc.:
14 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
Basis for effect:
other: Zooplankton eveness
14 wk
Dose descriptor:
Effect conc.:
21 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
Basis for effect:
other: chlorophyll a in periphyton
Details on results:
During baseline there were 20 genera of phytoplankton from six taxa.
• There were two major phytoplankton taxa during baseline and treatment: chlorophyta, cyanobacteria.
• For phytoplankton during treatment: evenness and diversity were adversely affected and although chlorophyta and cyanobacteria were the major
taxa the chlorophyta abundance significantly decreased and cyanobacteria increased. The lowest NOEC (14μg/L) was for chlorophyta evenness.

During baseline there were 34 genera of zooplankton from four taxa.
• For zooplankton during treatment: richness and diversity were not adversely affected but evenness was effected. Rotifer and copepod abundance increased and cladocera abundance decreased. Therefore, the lowest NOEC (14μg/L) was for evenness.

For chlorophyll a in periphyton the NOEC was 21μg/L.
Reported statistics and error estimates:
The study results were evaluated using the Dutch Guidelines for evaluating the effects in aquatic microcosm studies.
Treatment comparisons with the control for phytoplankton and periphyton chlorophyll a concentrations were performed using T-tests. Chlorophyll-a data were transformed using log-transformation if normal distribution and homogeneity of variance were not met. An effect with a p-value ≤ 0.05 was considered significant. Shannon diversity index (H = -Σ Pi(lnPi)) was used to determine diversity of zooplankton and phytoplankton in each microcosm (where Pi is the proportion of each species in the sample. Species richness (number of species per volume) and species abundance (number of organisms per volume) were also calculated for zooplankton and phytoplankton. Species richness and abundance for zooplankton are based on 1 L whereas for phytoplankton it is based on 1 mL. Differences in diversity, richness, and abundance between treatments were determined using analysis of variance (ANOVA) with alpha (α) of 0.05 for determination of statistical significance. All endpoints were determined using the measured time-weighted-average dissolved zinc concentration for each treatment.

Applicant's summary and conclusion

Validity criteria fulfilled:
Mesocosm/microcosm study done according to standard protocol and useful for validation the ecological relevance of laboratory generated ecotoxicity data. However, some inherent characteristics of the study (elevated T, derivation towards pH>9), result in very high sensitivity of the system to
Executive summary:

An extensive mesocosm/microcosm study has been performed to evaluate effects of a permanent zinc chloride exposure to an aquatic community in outdoor mesocosm/microcosms. The study is done according to the state-of-the-art protocols for substance and metal mesocosm/microcosm testing. The study included all important community elements (phyto and zooplankton, benthic invertebrates). Zinc was tested at permanent nominal concentrations of 8, 20, 40, 80, and 160 μg/L, which were maintained by spiking treatments every four days. Effects on community abundance, richness and diversity showed no relevant direct effects (NOEC) up to 14 μg/L. following 14 weeks of exposure. Some inherent characteristics of the study (elevated Temperature around 30°C, strong derivation of pH during the test towards values>9), are considered result in very high sensitivity of the system to zinc, and necessitate a careful interpretation of the study in a context of EU waters.